1. Field of the Invention
The present invention relates to a method for determining macromolecules such as proteins. The method comprises forming a stain image by nucleating polyacrylamide gels containing the macromolecules with a palladium tetramine salt and developing with a specific physical developer solution.
2. Description of Related Art
The detection of macromolecules such as proteins and polypeptides is extremely important in many areas of biology and clinical medicine such as genetic screening and the diagnosis of genetic diseases. In these areas, the amount of macromolecules that can be detected in cellular extracts of body fluids is determinative.
Electrophoresis is the movement of charged particles in a matrix under the influence of an electrical field. Electrophoresis is the primary laboratory detection and characterization technique for macromolecules (see Chemical and Engineering News 3/14/83, p. 10).
Continuing developments in two-dimensional gel electrophoresis have provided the capability of resolving thousands of macromolecules from complex biological mixtures. However, the inability to detect polypeptides present in low concentration has limited the application of this technology, particularly in clinical screening for pathological states, endocrinology, mammalian metabolism, developmental biology, and immunology.
Because the improved gel electrophoretic techniques greatly increase macromolecule resolution, visual detection methods employing conventional polypeptide dyes are no longer adequate.
The most commonly used conventional macromolecule stain is Coomassie Blue, which may be considered as a prototype. Dyes of this type are mainly dependent upon the electrostatic attraction between dye and polypeptide, stabilized by van der Waals forces. In fact, Coomassie Blue and a variety of other dyes exhibit particular affinities for macromolecules of specific charge. Coomassie Blue, an acidic dye, stains basic macromolecules most intensely, while crystal violet is the most effective stain for acidic macromolecules. Other dyes for which quantitative aspects of staining have been investigated include Amido Black, Fast Green, and Fe.sup.2+ -bathophenanthroline sulfonate. In contrast, the Remazol Brilliant Blue R method depends on a covalent bond between dye and polypeptide. With Coomassie Blue, the minimum amount of a protein that can be detected is approximately 0.2 to 0.5 .mu.g.
An assortment of other techniques also exist. These include densitometric scanning for absorbance at 280 nm, binding of radiolabelled or fluorescent ligands such as concanavalin A to glycoproteins, binding of antisera to polypeptides at the gel surface, and staining of specific polypeptide moieties.
Radioactive detection techniques offer a higher degree of sensitivity than the stains but are often impractical to use. In vivo radiolabelling may alter cellular metabolism and it may be impossible to label certain human macromolecules. In vitro radiolabelling has the disadvantage that it might alter the electrophoretic mobility of macromolecules. Furthermore, radioactive reagents sometimes prove too expensive and long exposure to detect trace macromolecules may result in the problem of "autoradiographic spreading".
The above staining methods, moreover, are difficult to perform, hazardous, time-consuming, and unless the macromolecules are heavily labeled, lack the sensitivity to detect proteins present in low or trace concentrations. A problem arises, for example, with body fluids, such as cerebrospinal and amniotic fluids, which are often difficult to obtain in quantity and frequently contain certain abundant proteins which cause distortion of electrophoretic patterns when sufficient sample is analyzed to observe specific trace polypeptides.
Another method of visualization involves the use of silver stains. In this method, a swollen polyacrylamide electrophoretic sheet is treated with a silver salt and subsequently developed in a dilute reducing solution such as formaldehyde. An improvement in sensitization is obtained as compared with dye staining. This method entails several washing steps and many of the solutions used are unstable and must be mixed immediately before use. The silver stain method is described in PCT application publication No. WO 82/03128, Sept. 16, 1982 by Merril.
There is a need for a macromolecule visualization procedure that achieves a higher detection sensitivity than obtained with dye staining, and which is similar and more expedient to carry out than is the case with dye and silver staining, or autoradiography.